The influence of 1% alpha-eleostearic acid (a-ESA, cis9,trans11,trans 13-18:3) and 1% punicic acid (PA, cis9,trans11,cis13-18:3) on fatty acid composition in mouse tissues was compared with conjugated linoleic acid (CLA, mixture of primarily cis9,trans11- and trans10,cis12-18:2) in the present study. The content (% total fatty acids) of 18:2n-6 was significantly reduced in the heart and adipose tissues, and total polyunsaturated fatty acids (PUFAs) and n-6 PUFA were significantly reduced in adipose tissue by a-ESA, PA and CLA feeding. The content of 22:6n-3 and total n-3 PUFA were significantly increased in the liver, kidney and heart by PA feeding, but not by a-ESA. In contrast to PA, supplementation with CLA significantly decreased 22:6n-3 in the liver, kidney and heart. The content of 20:4n-6 was significantly decreased in the liver and kidney by CLA feeding, but not by a-ESA and PA. The present results indicate that a-ESA, PA and CLA have differential effects on 22:6n-3 and 20:4n-6 content in mouse tissues. PRACTICAL APPLICATIONS Conjugated linolenic acid (CLnA), a group of octadecatrienoic acid isomers with a conjugated triene system, has been reported to exhibit favorable physiological effects, including anticancer properties and regulation of lipid metabolism. Punicic acid and alpha-eleostearic acid, two isomers of CLnA, have been shown to convert into cis9,trans11-18:2 in vivo. The effect of CLnA on fatty acid composition in mouse tissues was investigated in comparison with CLA mixtures in the present study. The data obtained here could provide information for the potential application of CLnA-containing seeds as functional food ingredients, a natural source of endogenously formed cis9,trans11-18:2 and a dietary feeding strategy to beneficially modify the fatty acid composition of animal tissues.